Note that the following discussion refers to a number of publications by author(s) and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-a-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes.
Photosynthetic plants utilize sunlight to power all cellular processes (directly or indirectly) and ultimately derive most or all of their biomass through chemical reactions driven by light. All commercially important photosynthetic plants belong to the kingdom Plantae. They include familiar organisms such as trees, herbs, bushes, grasses, vines, ferns, mosses, and the Chlorophytic green algae.
Plants obtain energy from sunlight via a process called photosynthesis. Photosynthesis is a process that converts carbon dioxide into organic compounds, especially sugars, using the energy from sunlight (Blankenship, 2010). Plants absorb sunlight via their photosynthetic antenna complexes, also called light harvesting complexes (LHC) and sometimes referred to herein as light harvesting antenna complexes, whose function it is to transfer excitation energy to the reaction center complexes of Photosystem II (PSII) and Photosystem I (PSI). The reaction centers then drive electron transfer, photophosphorylation and oxygenic reactions that lead to energy production and carbon capture in the form of complex biomolecules (e.g., sugar, starch, lipids, and the like).
Light harvesting antenna complexes for PSI (termed LHC I) and PSII (termed LHC II) are composed of pigment-protein complexes. Light harvesting antenna pigments including chlorophyll a (Chl a) and chlorophyll b (Chl b) and a variety of accessory pigments (e.g., carotenoids and xanthophylls) which participate in the complicated energy transfer route of photosynthesis. The PSII light harvesting complex includes the proximal (near) antenna Chl a binding proteins associated with the PSII reaction center; and the peripheral (distal) antenna Chl a, Chl b, and carotenoid binding proteins. The peripheral antenna complex of PSII (LHC II) further comprises the major (outer) more abundant trimeric antenna proteins that are encoded by nine genes (LHCBM 1-LHCBM9) and a core (inner) antenna protein complex that is encoded by three genes (LHCB4, LHCB5 and LHCB7) (Minagawa and Takahashi, 2004). LHC II proteins contain up to 80% of the total chlorophyll in plant and algal thylakoid membranes.
The process of photosynthesis has been optimized through evolution to produce plants adapted to be more fit in natural environments; it was not, however, necessarily optimized to give the highest harvest index in monoculture conditions relevant to agriculture. Here “harvest index” refers to the yield of the desired product compared to the total mass of the plant. Strategies involving reducing the optical cross-section of photosynthetic light-harvesting antenna size have been developed and successfully implemented in algal cultures, resulting in improved productivity. The instant invention describes a similar approach that can be also utilized in plants for improved productivity. Engineered plants with a range of chlorophyll a/b ratios, and, as a result, light harvesting antenna sizes can be produced. An optimal range of antenna sizes results in improved photosynthetic performance and the invention describes a sharp transition point where further reductions in antenna size becomes detrimental to photosynthetic efficiency. We hypothesize that this transition point is related to a phase transition in the arrangement of photosystem II in thylakoid membranes, regulated by the abundance of light harvesting complexes II. Plants with optimized antenna perform well not only in controlled greenhouse conditions, but also in the field. Embodiment of the present invention provides transgenic plants having improved yield of productivity, constructs and methods of use to produce said tranasgenic plants.
In nature, photosynthetic cells may adjust to altered light environments in order to optimize energy capture and conversion efficiency or, alternatively, to protect the cells from too much light. Cells adjusted to low light levels typically possess larger light harvesting antenna complexes than those acclimated to high light intensities so as to maximize light capture at limiting light conditions. Such low light acclimated plants have lower Chl a/b ratios as the photosystems contain relatively high amounts of Chl b and have a large light-harvesting complex (LHC) antenna (composed mostly of chlorophylls a and b). It has been reported plants that can adapt to high light intensity but the linkage to defined chl a/b ratios and improved yields has not been proven. Adapted plants that are grown under high light (HL) intensity, were shown to have relatively low amounts of Chl b in their LHC, smaller LHC antenna, and a higher Chl a/b ratio (Björkman et al., 1972; Leong and Anderson, 1984; Larsson et al., 1987). However, other studies have shown that photosynthetic cells acclimated to high light intensities have 50% lower cellular Chl contents but show only slight (if any) increases in the Chl a/b ratio (Neale and Melis, 1986).
A negative consequence of having efficient light harvesting complexes is that photosynthetic electron transfer in nearly all photosynthetic cells becomes light saturated at only 25% of full sunlight intensity (here full sun is assumed to be 2000 μmol photons m-2s-1 at 400-700 nm) (Polle et al., 2001). At high photosynthetic photon flux densities (photosynthetic photon flux density is a measure of the number of photons in the 400-700 nm range of the visible light spectrum that fall on a square meter of target area per second), the rate of photon absorption exceeds the rate at which photosynthesis can convert the energized antenna complexes into productive charge transfer processes (that are used to produce reducing equivalents and energy). Over excitation of the light harvesting antenna complex under high light intensities increases the potential for long-lived excited states and photo-oxidative damage in plants, this is due to the generation and accumulation of reactive oxygen species (ROS) such as chlorophyll in its triplet state (3Chl) and reactive oxygen species (Krieger-Liszkay et al., 2008, Vass and Cser, 2009).
Plants have short- and long-term responses to protect the photosynthetic apparatus from the harmful effects of excess light. Short-term responses include the thermal dissipation of excess absorbed photons (qE) and state transitions (qT), both of which are components of non-photochemical quenching (NPQ). The qE (energy-dependent quenching) processes involve the de-excitation of Chl in its singlet excited state (Chl*) formed in the PSII antenna upon light absorption to minimize the formation of triplet state chlorophyll (3Chl) and ROS in the photosynthetic apparatus (Muller et al., 2001). Processes associated with qT are involved in regulating the relative excitation of PSII and PSI and thereby regulate linear and cyclic electron flow during photosynthesis (Eberhard et al., 2008). Longer term responses occur over hours and days after high light exposure and include transcriptional and translation level changes in LHC mRNAs and high light induced mRNAs, PSII core protein (D 1) turnover and PSII repair, and increases in the xanthophyll cycle carotenoids. Hence, under high light intensities, up to 80% of absorbed photons can be dissipated as heat or fluorescence due to fluorescence and the activation of the short-term photoprotective responses (NPQ) causing large decreases in light utilization efficiency and photosynthetic productivities (Polle et al., 2002).
Chl b is synthesized from proto-Chl a by chlorophyll a oxygenase (CAO) which is sometimes referred to in the literature as chlorophyll b synthase. Either nomenclature applies to the instant invention. The overexpression of the Cao gene leads to the enhancement of Chl b biosynthesis in Arabidopsis and consequently to an enlargement of the PSII-associated peripheral antenna (Tanaka et al., 2001). Significantly, Chl b-less mutants (cbs-3) of a green alga, Chlamydomonas, have substantially elevated light-saturated photosynthetic oxygen evolution rates (up to 1.25 fold increase when expressed on a Chl basis) compared to the wild-type and do not light saturate at full sunlight intensities (Polle et al., 2000). Moreover, studies where the size of the LHC II has been preferentially attenuated have shown that reducing PSII antenna size (and not PSI) results in higher rates of oxygen evolution at high light intensities than wild-type cells (Polle et al., 2002; Polle et al., 2001).
Embodiments of transgenic plants of the present invention are engineered to artificially modulate the light harvesting complexes. For example, the modulation of the LHC can occur in a tissue-specific manner that is, the expression of one or more genes is linked either to a light-activated promoter or is driven by a tissue-specific promoter that is only active in photosynthetic tissues of the plant. Unexpectedly, a transgenic plant as described according to one embodiment of the present invention provides for improved partition of a significant amount of the improved carbon fixation into storage tissues such as the seed or starch rather than a generalized increase in biomass of all tissues. Additionally, these transgenic plants have improved rates of growth, starch accumulation in plastids, and non-photochemical quenching (high light photoprotection) in comparison to wild-type plants grown under the same condition.
This and other unmet needs of the prior art are met by exemplary compositions and methods as described in more detail below.